Summary

Recent work on species with simple leaves suggests that the juxtaposition
of abaxial (lower) and adaxial (upper) cell fates (dorsiventrality) in leaf
primordia is necessary for lamina outgrowth. However, how leaf dorsiventral
symmetry affects leaflet formation in species with compound leaves is largely
unknown. In four non-allelic dorsiventrality-defective mutants in tomato,
wiry, wiry3, wiry4 and wiry6, partial or complete loss of
ab-adaxiality was observed in leaves as well as in lateral organs in the
flower, and the number of leaflets in leaves was reduced significantly.
Morphological analyses and expression patterns of molecular markers for
ab-adaxiality [LePHANTASTICA (LePHAN) and LeYABBY B
(LeYAB B)] indicated that ab-adaxial cell fates were altered in
mutant leaves. Reduction in expression of both LeT6 (a tomato
KNOX gene) and LePHAN during post-primordial leaf
development was correlated with a reduction in leaflet formation in the wiry
mutants. LePHAN expression in LeT6 overexpression mutants
suggests that LeT6 is a negative regulator of LePHAN. KNOX
expression is known to be correlated with leaflet formation and we show that
LeT6 requires LePHAN activity to form leaflets. These
phenotypes and gene expression patterns suggest that the abaxial and adaxial
domains of leaf primordia are important for leaflet primordia formation, and
thus also important for compound leaf development. Furthermore, the regulatory
relationship between LePHAN and KNOX genes is different from
that proposed for simple-leafed species. We propose that this change in the
regulatory relationship between KNOX genes and LePHAN plays
a role in compound leaf development and is an important feature that
distinguishes simple leaves from compound leaves.

Several genes in several species are thought to specify the adaxial and
abaxial domains. For example, leaf adaxial cell fate is replaced by abaxial
cell fate in the phantastica mutation of Antirrhinum,
suggesting that PHANTASTICA (PHAN), a MYB domain
transcription factor, plays an important role in establishing (or maintaining)
adaxial cell fate in leaf primordia
(Waites and Hudson, 1995;
Waites et al., 1998). In
Arabidopsis ARGONAUTE1 (AGO), REVOLUTA (REV) and
PINHEAD (PNH) are also important for specifying adaxial cell
fate in lateral organs and for promoting meristematic activity in the SAM and
axillary meristems (Bohmert et al.,
1998; Lynn et al.,
1999; Talbert et al.,
1995). PHABULOSA and PHAVOLUTA are homeodomain-leucine zipper (HD
ZIP III) proteins with a START (steroid/lipid-binding) domain expressed in the
adaxial cells of the leaf primordium and in the SAM and semi-dominant
mutations in these genes produce radial leaves with adaxial cell fates
(McConnell and Barton, 1998;
McConnell et al., 2001). In
the leafbladeless mutant in maize, ectopic patches of abaxial
identity are seen on the adaxial side of the leaf and ectopic lamina forms at
the boundary between the two cell fates
(Timmermans et al., 1998).
FILAMENTOUS FLOWER (FIL), YABBY2 (YAB2),
YABBY3 (YAB3) and KANADI are expressed only
abaxially in all lateral organs of Arabidopsis, and ectopic
expression of FIL or YAB3 is sufficient to induce ectopic
abaxial patches in the adaxial region of the leaf
(Sawa et al., 1999a;
Siegfried et al., 1999).
Together, all these mutant phenotypes strongly suggest that the juxtaposition
of adaxial and abaxial cell fates is necessary for proper leaf lamina
development in simple-leafed species, and that adaxial and abaxial cell fates
are mutually antagonistic.

We describe four non-allelic mutants, wiry (w),
wiry3 (w3), wiry4 (w4) and wiry6
(w6) that are defective in ab-adaxial symmetry in tomato. The degree
of leaf compounding in these mutant plants was severely reduced. The
expression patterns of LeT6, TKN1, LePHAN and LeYAB B were
determined in the w, w3 and w6 mutants. The regulatory
relationship between LePHAN and KNOX genes in the meristem
and early leaf primordium is different from that seen during the later stages
of leaf development in tomato and may explain the compound nature of the
tomato leaf.

The w and w4 loci are on chromosome four (at 20 cM and 28
cM from the distal end of short arm). The w6 locus was mapped using
an F2 mapping population from a cross between w6 (L.
esculentum) and L. pennellii
(Tanksley et al., 1989). Using
recombination between the w6 mutant phenotype and a LePHAN
RFLP (HindIII) between L. esculentum and L.
pennellii, we determined that the w6 locus is 30 cM from the
LePHAN locus on chromosome 10.

Histology and scanning electron microscopy

Tissues for plastic sections were fixed and sectioned as described
previously (Kessler et al.,
2001). Samples were viewed with a Nikon Eclipse E600 microscope
and images collected using a SPOT (RT Color) digital camera. Samples for SEM
were fixed and viewed as described previously
(Kessler et al., 2001).
Electronic images, collected either directly from the SEM or from a SPOT
camera, were processed in Adobe Photoshop.

In situ hybridization and RT-PCR in situ hybridization

In situ hybridizations were performed as described previously
(Long et al., 1996) using
full-length cDNA probes for LeT6, TKN1, LeYAB B and LePHAN.
Approximately 500,000 pfu of a λgt10 library from 6-7 mm tomato flowers
were screened using INNER NO OUTER, a YABBY member, as probe
(Villanueva et al., 1999) to
obtain LeYAB B. Median sections (containing the SAM) from multiple
different tissue samples including positive controls were placed on each slide
and processed. Each experiment was repeated at least four times. Tissues for
RT-PCR in situ hybridizations were embedded, sectioned with a Zeiss Microtome
HM340E, and processed as previously described
(Long et al., 1996). Instead
of an overnight hybridization step, RT-PCR was performed on sections as
previously described (Ruiz-Medrano et al.,
1999). Primers used for the RT-PCR in situ experiments were
designed based on the cDNA sequence of LePHAN and LeT6 as
follows:

LePHAN1: 5′ACGAGCAGCGTCTTGTTATACAACTAC3′,

LePHAN2: 5′CCCTTCGTCTAAATCCTTGCAGC3′,

LeT65′: 5′TCTTTAACTAACAATAACAATGCAGAAAC3′,

LeT63′: 5′CCAAAGCAGATTCATGAGAAGAATAG3′.

Immunolocalization

Immunolocalization was performed as described previously
(Jackson et al., 1994) using a
polyclonal antibody against ROUGHSHEATH2 [a generous gift from Dr Marja
Timmermans, for details on antibody preparation see Kim et al.
(Kim et al., 2003)].

RESULTS

Abaxialization of leaf and reduction of leaflet number in w,
w3 and w6 plants

Wild-type tomato produces unipinnate compound leaves with 7-9 leaflets
(Fig. 1A). w, w6 and
w3 plants produced mostly cup-shaped or wire-like leaves, but
occasionally produced twisted, irregularly shaped flattened leaves with one or
two leaflets (Fig. 1B,C,E). In
the compound leaves of the w, w3 and w6 mutants, there were
27%, 34% and 19.9% leaflets respectively, compared to wild type (100%;
Table 1). The incidence of
cup-shaped or wire-like leaves increased in later stages of plant development.
A unique morphology was often seen in w3 leaves. These leaves
subtended an axillary bud. After production of one or two leaflet pairs, the
rachis split and each branch produced an almost complete compound leaf. Often
at the junction of the split an axillary-bud like structure was seen
(Fig. 1C,D). The w3
and w6 mutant plants produced cup-shaped leaves. In contrast,
w mutants made tendril-like terminal leaflets. The w, w3 and
w6 mutant plants formed normal axillary buds in the axils of the
wire-like leaves (Fig. 1F).

To determine if wire-like leaves were produced by abaxialization or
adaxialization, the anatomy of these leaves were examined. All parts of a
wild-type leaf (including petiole and rachis) have distinct ab-adaxiality.
Vascular bundles in the tomato leaf are amphiphloic with both abaxial and
adaxial phloem flanking the central xylem
(Fig. 2A). Elongated palisade
mesophyll cells are located in the adaxial side of the leaf and spongy
mesophyll cells are present in the abaxial region of the leaf lamina
(Fig. 2B). The w, w3
and w6 wire-like leaves were radially symmetric
(Fig. 2E-H). This anatomy
differed both from the wild-type stem, with a cylinder of vascular tissue
surrounding a central pith, and from the wild-type petiole, with clear
ab-adaxial symmetry (Fig.
2C,D). Vascular bundles of w, w3 and w6 leaves
often had xylem in the center encircled by phloem
(Fig. 2E,G,H). Mesophyll cells
surrounded the central solid vascular cylinder, but did not have features of
distinct elongated palisade mesophyll cells
(Fig. 2E-H). In w3
leaves producing ectopic leaves with axillary buds, the primary rachis, prior
to splitting, had an incompletely closed ring-shaped vascular bundle (arrow),
suggesting that this leaf is chimeric with features of both the leaf and the
stem (Fig. 2F).

The expanded and flattened leaves of w3 and w6 often
showed abaxial patches on the adaxial side of the leaf. In these abaxial
patches palisade mesophyll cells were replaced by spongy mesophyll cells
(Fig. 2I,J). The w3
and w6 leaf had a semicircular vascular bundle with the inner phloem
clustered at one end on the adaxial side
(Fig. 2L,M) rather than a
horseshoe-shaped vascular bundle in the midrib as in the wild-type leaf
(Fig. 2A). This suggests a
reduced adaxial domain in w3 and w6 leaves. The flattened
w leaves had normal mesophyll differentiation in the leaf lamina
(Fig. 2K). However, in
w, vascular bundles in the midrib were reduced and ectopic palisade
cells developed on top of the midrib region
(Fig. 2N).

Scanning electron microscopy (SEM) revealed that epidermal cell fates were
altered in w3 and w6 leaves. The adaxial epidermal cells of
the wild-type leaf were less lobed with fewer crenulations and very few
stomata (Fig. 3A), while the
abaxial epidermal cells were highly crenulated and irregularly zigzag-shaped
with lots of stomata (Fig. 3B).
In addition, the wild-type adaxial leaf surface was smooth, compared to the
rougher abaxial leaf surface. In contrast, both the upper and lower epidermal
cells of w3 leaves had characters intermediate between those seen in
the abaxial and adaxial surface of wild type. Both epidermal cells were less
lobed (like wild-type adaxial epidermal cells) and had more crenulations (like
wild-type abaxial epidermal cells) with roughly equal numbers of stomata,
suggesting the loss of distinct abaxial-adaxial epidermal differentiation
(Fig. 3C,D). In w6,
epidermal cells on both leaf surfaces were highly crenulated and irregular in
shape, suggesting abaxialization of the adaxial epidermis of the leaves
(Fig. 3E,F). However,
w epidermal cells were normal with distinct ab-adaxial features
(Fig. 3G,H).

Floral organs of w, w3 and w6 are
abaxialized

To see if other lateral organs were also abaxialized, we examined floral
organs in w, w3 and w6. Tomato flowers have five sepals,
petals and stamens and two fused carpels. The bases of sepals are fused into a
cup-shaped structure. The corolla is tubular and anthers are adnate to the
corolla tube (Fig. 4A). w,
w3 and w6 flowers usually had extra floral organs (e.g. 7-10
sepals and petals) and lacked the fusion of floral organs seen in normal
flowers. The lateral organs of w, w3 and w6 flowers were
narrower than those of wild type (Fig.
4B-D).

In wild-type petals, the adaxial epidermis has protruding papillar cells
and no trichomes, while the abaxial epidermis has flattened cells and many
trichomes (Fig. 4E,I,J).
Vascular bundle organization was not altered in the w6 petal
(Fig. 4F). However, papillar
cells of the w6 adaxial epidermis had interspersed trichomes.
Moreover, the boundary between papillar cells and flattened cells was moved
from the margin of the petal toward the adaxial side, indicating a reduction
in the adaxial domain of the w6 petal
(Fig. 4K). In w3
plants, petals were even more abaxialized than in w6 petals; both the
abaxial and adaxial epidermal surfaces of w3 petal had flattened
cells and numerous trichomes (Fig.
4L,M). The w3 petal had all inner cells packed tightly,
resembling the collenchyma cells of the midvein regions
(Fig. 4G). Frequently,
w petals were radially symmetric and among the papillar cells abaxial
patches of flattened cells were seen (Fig.
4H,N). A summary of wiry mutant phenotypes is given in
Table 2.

Tomato PHANTASTICA (LePHAN) expression is altered
in w, w3 and w6

phan mutant plants have a reduced adaxial domain in leaves. We
determined if the abaxialized phenotypes and reduced leaflet formation in
w, w3 and w6 are due to defects in PHAN expression.
Southern blot analysis showed one copy of LePHAN in the tomato genome
(data not shown). The chromosome location of LePHAN does not coincide
with that of w, w4 and w6. LePHAN mRNA expression was
determined by conventional in situ hybridization and by RT-PCR in situ
hybridization in the shoot apices and leaves of wild-type, w, w3 and
w6 plants.

In the wild-type apex, LePHAN mRNA levels were severalfold higher
in the leaf primordia than in the SAM central zone. During early leaf
development in wild type, LePHAN transcripts were detected in both
adaxial and abaxial sides of the leaf primordium
(Fig. 5A), but later, as the
leaf primordium grew out, LePHAN mRNA was confined to the adaxial
side (Fig. 5B). At later
developmental stages, strong LePHAN expression was detected in the
leaflet primordium and immature leaflet lamina regions
(Fig. 5C).

In the w6 SAM, reduced LePHAN expression was detected in
the central zone. Moreover, LePHAN expression in the early leaf
primordium, especially on the adaxial side, was much reduced compared to that
in wild type (Fig. 5G). No
LePHAN was detected in later stages of leaf development of wire-like
leaves, but the LePHAN transcript was detected in growing leaflet
primordia and leaflet blades in the developing cup-shaped leaves and in leaves
with reduced leaflet numbers (Fig.
5G inset). In w and w3 plants, no alteration of
LePHAN mRNA accumulation was detected in the SAM and early stage leaf
primordia, but LePHAN expression was absent in the later stages of
wire-like leaves (Fig. 5L,P and
insets).

LeYABBY B expression in w, w3 and
w6

To further characterize ab-adaxiality in w, w3 and w6
leaves, we examined the expression of LeYAB B, a member of the
YABBY gene family, in leaves of wild-type and the wiry mutants.
LeYAB B was expressed in the abaxial regions of wild-type leaf
primordia (Fig. 5D). In
w6 plants, LeYAB B expression was seen in both the adaxial
and abaxial sides of later radial leaves, and serial sections showed a hollow
tube-like pattern of expression (Fig.
5H,I arrow). Earlier flattened w6 leaves showed a
wild-type LeYAB B expression pattern
(Fig. 5H,I arrowheads).
Interestingly, in the w3 leaf primordium, LeYAB B mRNA
accumulated on the adaxial instead of the abaxial side
(Fig. 5M arrow). No LeYAB
B expression was detected in the w leaf primordium
(Fig. 5Q). These results were
confirmed by northern hybridization to RNA extracted from wiry shoot
apices (data not shown).

Expression of LeT6 and TKN1 in w,
w3 and w6

To determine if reduced leaflet formation in w3 and w6
leaves is due to the alteration of class I KNOX gene expression and
to determine the regulatory relationship between LePHAN and
KNOX genes in tomato, the mRNA expression patterns of two class I
KNOX genes, LeT6 (the tomato STM ortholog) and
TKN1 (the tomato KNAT1 ortholog) were examined.

In wild type, LeT6 mRNA accumulates in the SAM, in the early leaf
primordia, and later in leaflet primordia and growing leaflet blades
(Fig. 5E)
(Chen et al., 1997). Strong
LeT6 expression was detected in the central zone of wild-type SAM
(Fig. 5E). In the w
and w3 mutants less LeT6 mRNA was detected in the region of
the SAM (Fig. 5N,R).
Downregulation of LeT6 mRNA was seen in later stages of w,
w3 and w6 leaf development. This is equivalent to the stage
producing leaflet primordia and growing leaflet lamina in wild type. No
LeT6 mRNA could be seen in w6 plants that were producing
wire-like leaves (Fig. 5J
inset, R inset). However, LeT6 mRNA localized in the leaflet and
leaflet lamina regions of w, w3 and w6 plants that were
producing leaves that either were cup-shaped or had a reduced number of
leaflets (Fig. 5N insets).

TKN1 expression could be seen in the wild-type SAM, leaf primordia
and growing leaflet lamina, but the signal was stronger in the leaf primordia
and the peripheral zone of the SAM than in the central zone
(Fig. 5F). In w6
plants, a high level of TKN1 RNA was detected throughout the SAM and
in both early and late leaf primordia, including the radially symmetrical
primordia (Fig. 5K). In
w3 and w, TKN1 expression was normal in the SAMs but
downregulated in the leaflet primordia
(Fig. 5O,S). Expression of
TKN1 was absent at the tip of the leaf primordium (distal region),
where abaxialized wire-like structures are seen in w shoots
(Fig. 5S arrow).

The expressions of KNOX genes (LeT6 and TKN1)
were altered in wiry mutants. In particular, downregulation of
LeT6 in later stage of leaf primordia was accompanied by reduction of
leaf compounding in wiry mutants. Reduction of LePHAN
expression and upregulation of TKN1 in w6 suggests a
negative regulatory relationship between LePHAN and
TKN1.

LeT6 is a negative regulator of LePHAN in
tomato

To determine if LeT6 regulates LePHAN in tomato, we
analyzed LePHAN expression in Curl (Cu), a mutant
known to overexpress LeT6 (Parnis
et al., 1997). As reported
(Parnis et al., 1997), ectopic
expression of the LeT6 mRNA was detected in Cu leaflets and
leaflet lamina (Fig. 6A). In
Cu plants, LePHAN was present but reduced in the leaf
primordia, leaflet and leaflet blade regions
(Fig. 6B,C). This
LePHAN downregulation in Cu was not sufficient to cause a
LePHAN downregulation phenotype, as the Cu leaf showed
normal anatomy and epidermal cells (data not shown). Another LeT6
overexpression mutation, Mouse Ears (Me), is caused by a
gene duplication that leads to early overexpression of a homeobox-containing
fusion RNA (Chen et al., 1997;
Janssen et al., 1998;
Parnis et al., 1997). In the
Me mutant, LePHAN expression was reduced and the location of
expression was altered (Fig.
6E,F). In the Me plants, LePHAN expression was
reduced in the proximal region of the leaf primordia (data not shown) and
confined to a narrower adaxial domain in leaf primordia
(Fig. 6E). Often, LePHAN
expression was absent from the leaf primordia of Me/Me
(Fig. 6F), except in vascular
tissues, and the leaves produced were radial. This downregulation of
LePHAN correlated with the production simple leaves and wire-like
leaves at the upper nodes in Me/Me plants
(Fig. 6O), phenocopying
LePHAN downregulation phenotypes
(Fig. 6J). In these
LePHAN antisense transgenic plants, LePHAN expression was reduced to
a narrow domain or only to vascular tissues
(Kim et al., 2003), similar to
LePHAN expression in Me/Me
(Fig. 6F). Together, these data
suggest that LeT6 is a negative regulator of LePHAN.

LeT6 requires LePHAN activity in leaf
primordium

To determine if LeT6 expression is in turn regulated by
LePHAN, we made use of a LePHAN antisense transgenic line.
Several independent LePHAN antisense transgenic lines showed cup
shaped or wire-like leaves (Fig.
6J) and immunolocalization and in situ RT-PCR experiments showed
that LePHAN levels were reduced in these plants and petioles were
radial (Kim et al., 2003).

When Cu was crossed into a LePHAN antisense transgenic
line, the Cu phenotype was less severe, having less curled leaves and
often cup-shaped leaves with simple leaf blades
(Fig. 6H). The curled leaf
phenotypes were confined to distal region of the leaf. These plants showed
elongated and radially symmetric petioles
(Fig. 6G). These results
suggest that LePHAN downregulation phenotype is epistatic to
Cu and that the LeT6 overexpression phenotypes of
Cu require LePHAN activity. In Me/Me, LeT6
overexpression also led to the production of ectopic shoots on the leaves
(Fig. 6K,L, asterisk). These
ectopic shoots were formed only in the narrow adaxial domains, where
LePHAN was expressed (Fig.
6E). Often this adaxial domain converged to a point
(Fig. 6L) and ectopic shoots
emerged at this point, suggesting that LePHAN activity is required
for LeT6 overexpression phenotypes in Me.

Because a phenocopy of PHAN downregulation is seen only in
homozygous Me, but not in heterozygous Me plants, the
suppression of LePHAN by LeT6 seems to be dosage sensitive.
Me/+ plants show a typical KNOX overexpression phenotype
with an increase in leaf compounding (Chen
et al., 1997; Parnis et al.,
1997) (Fig. 6N).
Similarly, w6/w6 homozygous plants (with reduced LePHAN
levels) generated wire-like leaves (Fig.
6P) while, w6/+ heterozygous plants produced lobed
leaves, a phenotype also seen in the plants overexpressing KNAT1 in
Arabidopsis (Fig.
6P).

DISCUSSION

In w, w3 and w6 mutant plants, partial or complete
abaxialization of the lateral organs was observed throughout development,
suggesting that W, W3 and W6 play important roles in
establishing adaxial cell identity in all lateral organs. The tomato leaf
primordium develops basipetally (Dengler,
1984) and preferential distal abaxialization in the w
leaves indicates that W acts during early leaf development (in the
distal region), whereas W3 and W6 function later (in the
proximal region) in leaf development. Abaxialization of the wire-like leaf in
w, w3 and w6 is different from proximalization of the distal
region (changing blade domain into sheath domain) seen in maize mutants
(Becraft, 1994; Sinha and Hake,
1994; Tsiantis et al.,
1999) as unlike petioles, wiry leaves are radially symmetrical.
However, the occasional production of stem-like leaves that have ectopic
leaves with ectopic axillary buds (in w3) suggests that
proximalization into stem-like identity can occur in addition to
abaxialization. Adaxial cells are thought to be necessary for the induction of
axillary buds in Arabidopsis
(McConnell and Barton, 1998;
McConnell et al., 2001). The
abaxialized wire-like leaves of w, w3 and w6 formed normal
axillary buds on the adaxial side of the leaf base
(Fig. 1F). Sessile
(Arabidopsis) and petiolated (tomato) leaves may have different
potentials to form axillary buds in their leaf bases, and adaxial cell fate
may not be an absolute requirement for axillary bud formation in tomato. This
could also account for the presence of ectopic axillary buds in w3
mutant leaves.

It has been proposed that the boundary between abaxial and adaxial cell
fates is important for lateral lamina outgrowth
(Bowman et al., 2002;
Lynn et al., 1999;
McConnell and Barton, 1998;
Timmermans et al., 1998).
Reduced adaxial domain is accompanied by significantly reduced leaflet numbers
in w, w3 and w6 (Table
1). One explanation for the fewer leaflets in w, w3 and
w6 compound leaves is that leaflet primordium formation, like lamina
outgrowth, also requires a proper ab-adaxial boundary.

LePHAN expression in w, w3 and
w6

Two aspects of LePHAN expression in tomato set it apart from
orthologs in other species. No other PHAN ortholog has been reported
to be expressed in the SAM or specifically in the adaxial domain of leaf
primordia. At later stages of leaf development, LePHAN is expressed
only in the region of leaflet primordium initiation
(Fig. 5C), suggesting that
LePHAN (like LeT6) might be involved in leaflet formation,
or in establishing ab-adaxiality of leaflets. The possible function of
LePHAN in leaflet development is also supported by the fact that
LePHAN is not expressed in wire-like leaves and localizes to the
growing leaflet primordium or leaflet lamina region in cup-shaped, or less
compound leaves of w, w3 and w6. Downregulation of
LePHAN was seen in the leaf primordium and leaflet primordium in
these mutants, suggesting that W, W3 and W6 are positive
regulators of LePHAN expression in leaves. In addition, W6
may also regulate LePHAN expression positively in the meristem.

Regulatory relationship between LePHAN and KNOX
genes in tomato

Tomato LePHAN expression was reported to be absent from the SAM in
one study (Pien et al., 2001)
but was seen in the SAM and leaf primordia in a domain that overlaps the
KNOX expression domain by others
(Koltai and Bird, 2000). Our
results indicate that the latter is the case and that LePHAN
(Fig. 5A,
Fig. 6F,G) and TKN1
are expressed most strongly in the peripheral zone of the meristem, whereas
LeT6 expresses strongly in the central zone of the meristem
(Fig. 5A,E,F). In
Arabidopsis, STM is a negative regulator of AS1. This
regulatory relationship is conserved to a large extent in tomato. In
Cu and Me (LeT6 overexpression mutants),
LePHAN was reduced, suggesting that LeT6 is a negative
regulator of LePHAN. TKN1 was upregulated in w6 where
LePHAN was downregulated. A simple interpretation for the
upregulation of TKN1 in w6 is that LePHAN is a
negative regulator of TKN1. However, it is unclear how
LePHAN and TKN1 express in an overlapping manner in both the
SAM and early leaf primordia. Perhaps LePHAN and another gene (gene
A) have a mutually exclusive relationship and gene A in turn inhibits
TKN1 expression.

The regulatory dynamics between LePHAN, TKN1 and LeT6 in
later leaf and leaflet primordia is different from that in the meristem and
early leaf primordium. LePHAN, TKN1 and LeT6 all express in
the leaflet primordium and all of them are downregulated in the wire-like
leaves of w and w3. These expression data imply that the
negative regulation of LeT6 on LePHAN seen in the meristem
region does not hold in the wild-type leaflet primordium. Rather,
LePHAN functions with LeT6 in a coordinate manner.
Cu phenotypes were reduced in antiLePHAN/+ plants and
Cu and Me phenotypes were confined to the region where
LePHAN was expressed, suggesting that the LeT6
overexpression phenotype requires LePHAN function. Similarly,
downregulation of LePHAN masked TKN1 overexpression
phenotypes in w6/w6 and suggests that TKN1 also requires
sufficient LePHAN activity in the leaflet primordium in tomato.

LeT6 regulation of LePHAN is dosage sensitive

A reduced blade phenotype can be seen only in homozygous Me/Me
plants (Fig. 6O) and not in
heterozygous Me/+ plants (Fig.
6N), implying that a high dose of LeT6 is needed to
downregulate LePHAN in tomato. This hypothesis is also supported by
the fact that the expression domains where LePHAN and LeT6
express strongly do not overlap (Fig.
5A,E). We suggest that low levels of overexpression of either
LeT6 or TKN1 in leaf primordia can cause KNOX
overexpression phenotypes (such as increased dissection of leaves, or more
lobed or heart shaped leaves with palmate venation), but high levels of
LeT6 overexpression might lead to severe LePHAN
downregulation, causing a LePHAN downregulation phenotype. Thus,
w6/+ heterozygous plants produced highly lobed leaves
(Fig. 6P), a phenotype
generally attributed to KNOX gene overexpression, whereas
w6/w6 homozygous plants generated mostly cup-shaped or wire-like
leaves, which is a LePHAN downregulation phenotype
(Fig. 6P). Furthermore, this
LePHAN downregulation phenotype masks the KNOX
overexpression phenotypes in tomato, because a certain level of
LePHAN is required for the KNOX overexpression phenotypes
(as seen in Cu crossed to antiLePHAN and
Me/Me). This idea is supported by some of the phenotypes
seen in tomato plants that overexpress 35S::LeT6. Some
35S::LeT6 transgenic lines showed wire-like radially symmetrical
leaves, resembling PHAN downregulation phenotypes, instead of the
typical LeT6 overexpression phenotypes with more leaflets.
LeT6 overexpression was at much higher level in these plants
producing wire-like leaves, than in plants showing leaflet overproliferation
phenotypes (Janssen et al.,
1998).

We propose a model (Fig. 7)
that summarizes how LeT6 and LePHAN are regulated in tomato.
Our results suggest that LeT6 and LePHAN have a mutually
antagonistic expression pattern and that each is affected by the quantity of
the other. Thus, high levels of LePHAN repress LeT6 and
similarly high levels of LeT6 repress LePHAN. Our data does
not support increase in LeT6 expression by low levels of
LePHAN and vice versa. At intermediate levels both these genes
express. Since LeT6 is thought to be necessary for meristem formation
in higher plants (although this has not been directly demonstrated in tomato),
loss of LeT6 gene function or downregulation of LeT6 could
be lethal for plants. Low transformation and plant regeneration success in
experiments using 35S::LePHAN constructs support this hypothesis (our
unpublished data). LePHAN and LeT6 levels are well balanced
in the wild-type leaf, producing 7-9 leaflets with normal ab-adaxiality. Weak
LeT6 overexpression and LePHAN downregulation lead to
LeT6 overexpression phenotypes seen in the 35S::LeT6 plants
(Janssen et al., 1998),
Me/+ and Cu leaves. We suggest that the as1
mutation showing only KNOX overexpression phenotypes in
Arabidopsis and the rs2 phenotype in maize can be
categorized in this group. Perhaps, in these instances, KNOX
overexpression does not reach a level that would cause leaf lobing or the
PHAN downregulation phenotype. Strong KNOX overexpression
and LePHAN downregulation cause LePHAN downregulation
phenotypes including cup-shaped or wire-like leaves, as seen in the
as1 strong allele (Sun et al.,
2002), severe 35S::LeT6, Me/Me, w6/w6 and
Cu/Cu;antiLePHAN/+ leaves. However, it should be emphasized that a
direct interaction between the KNOX genes and PHAN has not
been proved and this interaction may involve multiple regulatory steps.

A model showing the regulatory relationship between LeT6 and
LePHAN and final leaf morphology in tomato. LeT6 is
downregulated when LePHAN is strongly overexpressed. Loss of
KNOX gene function or extreme downregulation of LeT6 could
be lethal for plants because of the lack of SAM formation/maintenance. Weak
LePHAN overexpression might lead to the ectopic leaf blade outgrowth
in the rachis region and make large simple leaves. LePHAN and
LeT6 levels are well balanced in the wild-type leaf, producing 8-9
leaflets with normal ab-adaxiality. Weak LeT6 overexpression and
LePHAN downregulation lead to KNOX overexpression phenotypes
seen in the 35S::LeT6 plants
(Janssen et al., 1998),
Me/+ and Cu leaves. Because LeT6 overexpression
phenotypes require LePHAN activity, strong LeT6
overexpression and LePHAN downregulation cause LePHAN
downregulation phenotypes including cup-shaped or wire-like leaves, severe
35S::LeT6, Me/Me, w6/w6 and Cu/Cu;antiLePHAN/+ leaves.

LeYAB B expression in w, w3 and w6
meristems

Our results show that, as seen for the Arabidopsis YAB3 gene,
LeYAB B is a good marker for abaxial cell fates
(Fig. 5D). LeYAB B
mRNA was detected throughout w6 leaf primordia
(Fig. 6H, I), while
LePHAN mRNA was downregulated in the adaxial region of the leaf
(Fig. 5G). This suggests that
adaxial cells of leaf primordia in w6 are converted into abaxial
cells. These results are consistent with the complete abaxialization of
adaxial cells of the w6 leaf (Fig.
2G). However, LeYAB B was unable to downregulate
LePHAN in the adaxial region of w3 leaf primordium
(Fig. 5L,M) and the absence of
LeYAB B did not cause ectopic expression of LePHAN in
w (Fig. 5P,Q). In
w3, LeYAB B was expressed in the adaxial region of the lateral organs
(Fig. 5M). However, in
w3, the absence of LeYAB B in the abaxial region still
allowed cells to have abaxial fate. While, presence of LeYAB B in the
adaxial domain did not cause complete abaxialization, the adaxial epidermis
attained some abaxial features (Fig.
3C), suggesting that LeYAB B may play a role in the
acquisition of abaxial cell fates. yab and fil mutants have
been reported to upregulate KNOX gene expression and result in
ectopic shoots in Arabidopsis
(Kumaran et al., 2002). By
contrast in tomato, ectopic expression of LeYAB B in the adaxial
region and absence in the abaxial region of the w3 leaf accompanies
ectopic bud formation in these leaves. The fact that ectopic expression of
LeYAB B in the adaxial region was detected in both w3 and
w6 leaf primordia, but LePHAN was downregulated only in
w6 leaf primordium, and complete abaxialization of adaxial cells was
seen only in w6 leaf all suggest that LePHAN and other
adaxial specific genes play a major role in controlling ab-adaxiality, while
the YABBY genes might be involved in a downstream part of the cell
fate acquisition pathway. Our results suggest that KNOX gene
expression is regulated by presence or absence of LePHAN and not
LeYAB B in tomato.

Is a compound leaf a reiterated shoot system or a carved simple
leaf?

The origins and homologies of compound leaves have been a matter of debate.
One view is that dicot compound leaves are a homeotic reiteration of simple
leaves along the rachis region of a compound leaf
(Lacroix and Sattler, 1994;
Rutishauser, 1995). In
contrast, others proposed that a compound leaf is formed by dissecting or
carving a simple leaf, perhaps by inhibition of blade formation in the rachis
area (Hagemann, 1984;
Kaplan, 1975).

The adaxial domain is necessary for leaflet primordium formation in tomato.
This is reminiscent of the situation where the adaxial domain of a leaf
primordium is required for normal SAM activity, and is suggestive of some
similarity between compound leaves and shoot systems. This similarity is
further supported by the presence of KNOX gene expression in the
leaflet primordia in all compound-leafed species from ferns and cycads to
higher plants (Bharathan et al.,
2002).

If the expression of KNOX genes is crucial to make compound
leaves, introducing the expression of KNOX genes into the leaf would
have been an important evolutionary innovation that led to the occurrence of
compound leaves. In Arabidopsis, Antirrhinum and maize (simple
leaves), no KNOX gene expression can be seen in the leaf primordia at
any stage of leaf development. Perhaps this is due to the fact that
PHAN and KNOX have a very tight mutually exclusive
regulatory relationship in Arabidopsis, Antirrhinum and maize
(Byrne et al., 2000;
Schneeberger et al., 1998;
Timmermans et al., 1999;
Tsiantis et al., 1999;
Waites et al., 1998). Our
study shows that both KNOX (LeT6 and TKN1) and
LePHAN are expressed in leaflet primordia, suggesting that
KNOX genes and LePHAN are not mutually exclusive in the
tomato leaflet primordium and that their functions might be dependent on each
other. Acquisition of a positive regulatory relationship between KNOX
genes and LePHAN in the leaf primordium might be an evolutionarily
significant change to introduce leaflet formation in the ancestral simple leaf
primordium. In fact, the discovery that the regulation between KNOX
genes and LePHAN of tomato differs from that of simple-leafed species
raises several questions. It will be interesting to determine if the positive
regulatory relationship between KNOX genes and PHAN is
conserved among compound-leafed species and if this positive regulation is
responsible for allowing KNOX expression in leaf primordia of
compound-leafed species.

Acknowledgments

The authors thank Drs John Harada, Connie Champagne and Sharon Kessler for
critical comments; members of the Sinha lab for advice and help; members of
the Rost Lab for help with histology; Dr Nancy Dengler for many helpful
discussions; the Charles M. Rick Tomato Genetics Resource Center for seed
stocks; and Dr Marja Timmermans for the anti-RS2 antibody. This paper is
dedicated to the late Dr Charles M. Rick for inspiring us to study the tomato
leaf. This work was supported by NSF IBN-9983063 and IBN-0316877 to N.S.;
Elsie Stocking Memorial, Rosalinde Russell, and UC Davis Jastro-Shields
Research Fellowships to M.K.; and SHARP (Howard Hughes) Fellowship to T.P.

References

Barton, M. K. and Poethig, R. S. (1993).
Formation of the shoot apical meristem in Arabidopsis thaliana: an
analysis of development in the wild type and in the shoot
meristemless mutant. Development119,823
-831.

Similar articles

Other journals from The Company of Biologists

“It really is a core responsibility of scientists … to be willing to speak out on behalf of their science”

George Daley, who was awarded the ISSCR 2017 Public Service Award, talks about his career investigating the biology and clinical application of stem cells, the importance of ethical guidelines and of science advocacy.

The 2017 EMBO/EMBL Symposium ‘Metabolism in Time and Space’ was held at EMBL Heidelberg, Germany, in May 2017. Alena Krejci and Jason M. Tennessen review this interdisciplinary meeting that provided a valuable forum to explore the interface between developmental biology and metabolism.

Articles of interest in our sister journals

In this Research article published in Journal of Cell Science, Qiang Wang and colleagues show that Net1 associates with Smad2 in the nucleus in a GEF-independent manner and facilitates Smad2 transcriptional activity to guide mesendoderm development in zebrafish.